The present invention relates to a method for the detection of telomerase activity, a method for the detection of cancer cells, a method for the diagnosis of cancer, as well as a kit for the detection of cancer cells and/or the diagnosis of cancer.
Human somatic cells have 22 pairs of autosomes and a pair of sex chromosomes. A total of 46 chromosomes are present in a cell. These chromosomes are independent of each other and rarely associated with each other. Telomeres are responsible for this important function and telomerase plays a role in maintaining the presence of telomeres. Telomeres are located in the terminal end of chromosomes and, in human, have a characteristic sequence of a few hundreds of repeated 6 bases 5xe2x80x2-TTAGGG-3xe2x80x2. The replication of DNA is essential to proliferation of cells by division and, based on the mechanism of the DNA replication, the terminal telomere of a chromosome is shortened once DNA has been replicated. Whenever the cell division is repeated, the telomeric sequence is shortened. Deletion of this portion induces association of chromosomes which causes adverse effects on cells. Further, other gene abnormalities may also occur and cells are led to death (senescence and death of cells). Telomeric sequences have an important role in the senescence and death of cells, that is, alternation of generations of cells. However, it is considered that if telomeric sequences of some cell populations which should naturally die (aged cell populations in which gene abnormalities have been accumulated) are still continuously added by the action of telomerase, a part of the populations may be immortalized and eventually cancerized. Accordingly, the detection of telomerase activity is very useful in the diagnosis of cancer or in monitoring the prognosis of treatment.
Recently, TRAP (telomeric repeat amplification protocol) for detecting the telomerase activity with a high sensitivity by using PCR (polymerase chain reaction) has been developed: Kim N. W. et al., (1994) Science, 206, 2011-2015; Piatyszek M. A. et al., (1995) Meth. Cell Sci., 17, 1-15. This method involves the detection of telomerase by a single primer extension assay system and is roughly divided in three steps. First, telomerase is extracted from cells. Then, extension reaction of TTAGGG chain by the telomerase is carried out and the reaction products are amplified by PCR using two primers, called TS and CX primers. Finally, the amplified products are electrophoresed to detect the telomerase activity by confirming ladders in autoradiography. This method has improved the detection sensitivity, enabling the detection of telomerase activity even in a small number of cells, such as 10 cells.
Among the above three steps, however, the detection system must involve electrophoresis, which requires complicated operations and a long period of time. For example, analysis of 32P- or fluorescence-labelled reaction products by polyacrylamide gel electrophoresis, HPLC, or other means is still required, so that the number of samples to be detected is limited and, in the case of 32P, its handling, such as treatment of gel or a large amount of waste liquid, is not easy. In addition, it will take a long time to conduct a series of operations, such as preparation of gel, electrophoretic separation (analysis) and exposure (detection), usually 2 to 48 hours.
These problems are very inconvenient, especially in the diagnosis of progression and prognosis of cancer which requires real time analysis, and it is also difficult to analyse a large amount of samples. Thus, there is a need for the development of another detection system which may replace the electrophoresis or autoradiography.
It is an object of the present invention to provide a method enabling rapid detection of telomerase activity with a high sensitivity.
The present inventors have eagerly studied the above problems and, as a result, found that telomerase activity can be rapidly detected with a high sensitivity by constructing a system comprising the DNA extension reaction with a teromerase in combination with the hybridization protection assay (HPA) developed by Gen-Probe, leading to the completion of the present invention.
That is to say, the present invention is a method for the detection of telomerase activity comprising amplifying an oligonucleotide sequence extended by a DNA extension reaction with a telomerase and hybridizing the resulting amplified product with a probe labelled with a non-radioactive labelling material to detect the telomerase activity. The non-radioactive labelling material includes, for example, acridinium esters, luminol, isoluminol, pyrogallol, protohemin, aminobutylethyl-n-isoluminol, aminohexylethyl-n-ethyl-isoluminol, and acridine derivatives.
The acridine derivatives may include, for example, those represented by the following formula I: 
wherein X denotes a halogen, or a group represented by the following formula II: 
in which X1 is a nitrogen, phosphorus, boron or arsenic atom, R1 is an alkoxy or aryloxy group, or a substituted or unsubstituted alkyl, alkenyl or aryl group, and R2 is a hydrogen atom, or, an alkoxy or aryloxy group, or a substituted or unsubstituted alkyl, alkenyl or aryl group, or the following formula III:
xe2x80x94X2xe2x80x94R2xe2x80x83xe2x80x83(III) 
in which X2 is an oxygen or sulfur atom and R2 is as defined above, Y denotes an oxygen or sulfur atom, or NH, R3 denotes a hydrogen atom, an amino, hydroxy, thiol, carboxylic acid, halogen, nitro, alkoxy or aryloxy group, or a substituted or unsubstituted acetyl, alkyl, alkenyl or aryl group, R4 denotes a substituted or unsubstituted alkyl, alkenyl or aryl group, and at least one of R1, R2, R3 and R4 comprises a reactive site capable of chemical bonding. The reactive site capable of chemical bonding herein means a site which binds to a probe. For example, when the bonding is effected through a linker and an amino linker having an amino group at the end thereof, said reactive site is a site which binds to the amino group. The substance capable of binding to this site includes, for example, a carboxylic acid derivative, preferably an acid halide or ester.
However, the present invention is not limited to the non-radioactive labelling materials described above. In addition to the acridine derivatives represented by the above formula I, those represented by the chemical formula I as shown in Japanese Patent No. 2602315 are also encompassed.
The oligonucleotide sequence extended through DNA extension reaction by a telomerase is amplified by a polymerase chain reaction using an oligonucleotide primer comprising at least a base sequence represented by xe2x80x9cAGNGTTxe2x80x9d wherein N is A, T, G or C at the 3xe2x80x2 side and/or an oligonucleotide primer comprising the base sequence represented by SEQ ID NO: 21. This amplification may also be effected by RNA synthetic reaction using an oligonucleotide primer comprising at least a base sequence represented by xe2x80x9cAGNGTTxe2x80x9d wherein N is A, T, G or C at the 3xe2x80x2 side and/or an oligonucleotide primer comprising the base sequence represented by SEQ ID NO: 21. A promoter sequence is added to at least one of oligonucleotide primers used in the RNA synthetic reaction.
Further, the amplification may also be carried out by polymerase chain reaction or RNA synthetic reaction using an oligonucleotide primer having any sequence which does not hybridize with the sequence represented by xe2x80x9cTTAGGGxe2x80x9d and has been added to the 5xe2x80x2 side of either an oligonucleotide primer comprising the base sequence represented by SEQ ID NO: 21 or an oligonucleotide primer comprising a base sequence having at least one nucleotide deleted, substituted or added in the base sequence of said primer.
In the present invention, the conditions under which no hybridization occurs may include, for example, 30 to 120xc2x0 C., preferably 37 to 90xc2x0 C.
The oligonucleotide primer comprising at least a base sequence represented by xe2x80x9cAGNGTTxe2x80x9d wherein N is A, T, G or C at the 3xe2x80x2 side includes that represented by SEQ ID NO: 1, and the oligonucleotide primer comprising the base sequence represented by SEQ ID NO: 21 includes that represented by SEQ ID NO: 2. Further, the promoter may include T7, T3 and SP6 RNA polymerase promoters.
Further, the present invention is a method for the detection of cancer cells comprising detecting the telomerase activity in the cancer cells by said method for the detection of the telomerase activity.
The cancer cells may include those contained in specimens obtained invasively or non-invasively. Specimens obtained invasively may include urinary bladder tissues, prostate gland tissues, uterus tissues, uterocervix tissues, udder tissues, pancreas tissues, liver tissues, large intestine tissues, stomach tissues, lung tissues, peripheral blood cells, kidney tissues, skin tissues, esophagus tissues, brain tissues and oral cavity tissues. Specimens obtained non-invasively may include urine, prostate gland juice, urinary bladder wash, uterus smear, pancreatic juice, duodenum juice, feces, oral cavity wash, enteron wash, saliva and sputum.
Further, the present invention is a method for the diagnosis of cancer comprising detecting cancer cells by said method for the detection of the cancer cells. The cancers include urinary bladder cancer, prostate gland cancer, uterus cancer, uterocervix cancer, breast cancer, pancreatic cancer, liver cancer, large intestine cancer, gastric cancer, lung cancer, kidney cancer, skin cancer, oral cavity cancer, esophagus cancer, brain tumor and leukemia.
Moreover, the present invention is a diagnostic kit comprising an oligonucleotide primer comprising at least a base sequence represented by xe2x80x9cAGNGTTxe2x80x9d wherein N is A, T, G or C at the 3xe2x80x2 end, an oligonucleotide primer comprising the base sequence represented by SEQ ID NO: 21 or an oligonucleotide primer comprising a base sequence having at least one nucleotide deleted, substituted or added in the base sequence of the primer, and a probe labelled with a non-radioactive labelling substance. Said kit also comprises a promoter added to at least one primer. Examples of the promoter are as above listed. Said diagnostic kit further comprises an oligonucleotide primer having any sequence which does not hybridize with the sequence represented by xe2x80x9cTTAGGGxe2x80x9d and has been added to the 5xe2x80x2 side of either an oligonucleotide primer comprising the base sequence represented by SEQ ID NO: 21 or an oligonucleotide primer comprising a base sequence having at least one nucleotide deleted, substituted or added in the base sequence of said primer.
Said diagnostic kit is used in the detection of telomerase activity or the diagnosis of cancer. Cancers to be diagnosed include urinary bladder cancer, prostate gland cancer, uterus cancer, uterocervix cancer, breast cancer, pancreatic cancer, liver cancer, large intestine cancer, gastric cancer, lung cancer, kidney cancer, skin cancer, oral cavity cancer, esophagus cancer, brain tumor and leukemia.
Further, the present invention is a method for the detection of telomerase activity comprising amplifying an oligonucleotide sequence extended through DNA extension reaction by a telomerase and detecting the resulting amplified product without using a radioactive labelling substance.
Hereinafter the present invention will be described in detail.
The present invention is a method for the detection of telomerase activity in cells or cell extracts (hereinafter referred to xe2x80x9ccell extractsxe2x80x9d). The present invention is to detect the telomerase activity using as a measure or index the presence or absence of an oligonucleotide sequence which is extended by the DNA extension reaction by a telomerase, and characterized in said oligonucleotide sequence is amplified and the amplified product is hybridized with a probe labelled with a non-radioactive labelling substance.
(1) Preparation of Cell Extracts and Amplification of Telomeric Repeat Sequences
First, cell extracts containing a telomerase are prepared from a cancer tissue or cell line.
The kind of cancer tissues or cell lines is not particularly limited but includes cancer tissues such as large intestine and liver cancers, as well as cancer cell lines of large intestine cancer, liver cancer, uterocervix cancer, chronic myelogenous leukemia, glioblastoma, breast cancer, fibrosarcoma or the like, for example, K562, MKN1, HeLa, U937, U373MG, T98G, A172, MCF-7, HT-1080, LoVo, WiDr, SW857 and VA-4.
The cell extraction may be carried out by any known method with or without some modifications: Kim N. W. et al., (1994) Science, 206, 2011-2015.
Then, an oligonucleotide primer comprising at least a base sequence xe2x80x9cAGNGTTxe2x80x9d wherein N is A, T, G or C at the 3xe2x80x2 side, hereinafter sometimes referred to as xe2x80x9cprimer 1xe2x80x9d, (for example, SEQ ID NO: 1) is added to the cell extract and the extension reaction of DNA is carried out. Preferably, the base sequence xe2x80x9cAGNGTTxe2x80x9d is desinged and synthesized such that it is 3xe2x80x2 terminal end of said primer 1. The length of said primer 1 is not particularly limited and the primer may be arbitrarily designed so long as at least base sequence xe2x80x9cAGAGTTxe2x80x9d, xe2x80x9cAGTGTTxe2x80x9d, xe2x80x9cAGGGTTxe2x80x9d or xe2x80x9cAGCGTTxe2x80x9d is contained at the 3xe2x80x2 side. For example, the length of the sequence is preferably 6 to 100 bases, more preferably 11 to 60 bases.
The DNA extended by the telomerase extension reaction (telomeric repeat sequence) is then amplified. The telomeric repeat sequence may be amplified by, for example, the polymerase chain reaction or RNA synthesis method. By using the polymerase chain reaction or RNA synthesis method, detection results may be obtained with a high sensitivity.
When the polymerase chain reaction is carried out, said primer 1 and/or, eg., an oligonucleotide primer comprising the base sequence represented by SEQ ID NO: 21, hereinafter also referred to as xe2x80x9cprimer 2xe2x80x9d (for example, SEQ ID NO: 2) is used as a primer. These primers may be chemically synthesized using a commercially available DNA synthesizer.
The polymerase chain reaction is carried out in a conventional assay buffer. A desired DNA may be obtained in a large amount by the polymerase chain reaction.
When the telomeric repeat sequence is amplified using the RNA synthesis method, the amplification may be carried out by, for example, the known procedures called TMA (Transcription Mediated Amplification) method (Japanese Patent Application Laying Open No. 4-500759).
Preferably, said TMA method is modified when used to amplify the sequence. Thus, a promoter sequence (hereinafter referred to as xe2x80x9cpromoterxe2x80x9d) is added to the 5xe2x80x2 end of the primer 1 as shown in FIG. 1 and DNA extension reaction is carried out using a telomerase as shown in FIG. 1 (1). The promoter may include, for example, T7 polymerase promoter such as SEQ ID NOs: 13 to 19, T3 polymerase promoter such as SEQ ID NO: 20, and SP6 polymerase promoter. However, the present invention is not limited to the promoters represented by said SEQ ID NOs.
Then, a reverse primer is used to convert the DNA into a double strand as shown in FIG. 1 (2). The reverse primer used herein is an oligonucleotide primer used to convert the .single stranded DNA extended by a telomerase into a double strand. As the reverse primer, for example, said primer 2 may be used.
From the double stranded DNA, a polymerase corresponding to said promoter is used to synthesize an RNA. For example, when T7 polymerase promoter, T3 polymerase promoter or SP6 polymerase promoter is used, T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase, respectively, is used to synthesize the RNA: FIG. 1 (3).
In the present invention, however, either one or both of the primers 1 and 2 may be used to carry out the RNA synthesis reaction. When either one of said primers is used, a promoter is added to the 5xe2x80x2 end of said primer to carry out the RNA synthesis reaction. When both of said primers are used, a promoter(s) may be added to the 5xe2x80x2 end(s) of the either one or both of said primers to carry out the RNA synthesis reaction.
The resulting RNA is subjected to DNA extension reaction using a reverse primer such as primer 2 to form a hybrid of DNA and RNA as shown in FIG. 1 (4). The RNA is decomposed and the DNA is extended to yield a double stranded DNA. This double stranded DNA fragment is used as a template to synthesize RNA using the above described various polymerases as shown in FIG. 1 (6). These reactions as shown in FIG. 1 (3) to (6) are repeated to symthesize in a large amount (amplify) the RNA. To stop the reaction, the reaction mixture is heated to, for example, 60xc2x0 C. In these procedures, the desired RNA may be obtained in a large amount.
Alternatively, instead of the reverse primer used in said modified TMA method, an oligonucleotide obtained by adding any sequence, that is, a base sequence randomly selected and synthesized so that it does not hybridize to the base sequence represented by xe2x80x9cTTAGGGxe2x80x9d, to the 5xe2x80x2 end of said reverse primer, may be used: refer to FIG. 1 (2). In the present invention, the oligonucleotide sequence randomly selected and synthesized is referred to as a tag sequence and a reverse primer to which a tag sequence is linked is referred to as a tag-sequence reverse primer. The tag sequence may also be linked to the full length reverse primer or a primer having a part (1 to 8 bases, preferably 4 to 6 bases) deleted, substituted or added in the 5xe2x80x2 or 3xe2x80x2 end of the reverse primer. Further, the tag sequence should be synthesized so that it does not hybridize to the base sequence represented by xe2x80x9cTTAGGGxe2x80x9d. Thus, the tag sequence may be partially complementary to the base sequence represented by xe2x80x9cTTAGGGxe2x80x9d so long as it does not hybridize thereto, or may also be unrelated to the base sequence represented by xe2x80x9cTTAGGGxe2x80x9d. The length of such a tag sequence may be 1 to 40 bases, preferably 5 to 30 bases in view of high amplification efficiency. The conditions under which hybridization does not occur may be, for example, 30 to 120xc2x0 C., preferably 37 to 90xc2x0 C.
(2) Detection of Telomerase Activity
To detect the oligonucleotide (DNA or RNA) thus obtained in a large amount, the present invention utilizes the hybridization protection assay (HPA) method developed by Gen-Probe Inc.: Japanese Patent Application Laying Open No. 2-503147.
The HPA method involves the use of an oligomer labelled with a non-radioactive labelling substance as a probe and the detection of chemiluminescence from said non-radioactive labeling substance when said probe hybridizes to the DNA or RNA to be detected. The characteristic of this method is to selectively hydrolyze the labelling substance of free probes to deactivate the labelling substance, rather than the physical separating operation such as washing carried out to distinguish hybridized probes from non-hybridized free probes. Accordingly, a desired target (nucleic acid or oligonucleotide) can be detected by simple operations within a short period of time.
The non-radioactive labelling substance may include, for example, acridinium esters, hereinafter referred to as xe2x80x9cAExe2x80x9d, luminol, isoluminol, pyrogallol, protohaemin, aminobutylethyl-n-isoluminol, aminohexylethyl-n-ethyl-isoluminol, and acridine derivatives.
The acridine derivatives may include, for example, those represented by the following formula I: 
wherein X denotes a halogen, or a group represented by the following formula II: 
in which X1 is a nitrogen, phosphorus, boron or arsenic atom, R1 is an alkoxy or aryloxy group, or a substituted or unsubstituted alkyl, alkenyl or aryl group, and R2 is a hydrogen atom, or, an alkoxy or aryloxy group, or a substituted or unsubstituted alkyl, alkenyl or aryl group, or the following formula III:
xe2x80x94X2xe2x80x94R2xe2x80x83xe2x80x83(III) 
in which X2 is an oxygen or sulfur atom and R2 is as defined above, Y denotes an oxygen or sulfur atom, or NH, R3 denotes a hydrogen atom, an amino, hydroxy, thiol, carboxylic acid, halogen, nitro, alkoxy or aryloxy group, or a substituted or unsubstituted acetyl, alkyl, alkenyl or aryl group, R4 denotes a substituted or unsubstituted alkyl, alkenyl or aryl group, and at least one of R1, R2, R3 and R4 comprises a reactive site capable of chemical bonding. However, the present invention is not limited to the above described non-radioactive labelling substances.
The halogen includes, for example, fluorine, chlorine, bromine, iodine and astatine. The alkyl includes those having 1 to 20 carbon atoms, preferably 1 to 5 carbon atoms, such as methyl, ethyl, propyl, butyl and amyl. The alkenyl includes those having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, such as vinyl and allyl. The aryl includes, for example, phenyl, tolyl, naphthyl and xylyl. The alkoxy includes those having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, such as methoxy and ethoxy. The aryloxy includes, for example, phenoxy and naphthoxy.
The probe used in the present invention is not particularly limited so long as it is extended through the telomerase extension reaction and is complementary to DNA or RNA amplified therefrom. Generally, the probe used comprises 10 to 40 bases, preferably 15 to 30 bases. The oligonucleotide used in the probe may be synthesized by the conventional phosphoamidite method using a commercially available DNA synthesizer. An amino linker for labelling DNA with AE or other labelling substance has been introduced during the chemical synthesis.
The DNA probe is labelled with AE or other labelling substance, for example, by reaction of the amino linker introduced during the DNA synthesis with an N-hydroxysuccinimide ester of AE.
The labelling site of AE or other labelling substance may vary freely depending upon the position of the amino linker introduced during the DNA synthesis: Yukio Matsuoka, et al., Clinical Pathology, Special Edition, Vol. 85, pp. 82-91 (1990).
i) Detection Using AE
As shown in FIG. 2, when the AE-labelled probe hybridizes to the amplified DNA or RNA (target), the AE is stabilized between the double helix of DNA. Even if hydrolysis is carried out for a given period of time, the ester linkage of AE is protected. Therefore, upon addition of an alkali and hydrogen peroxide, the acridinium ester is capable of generating chemiluminescence, from which the target can be detected: FIG. 2 (1).
On the other hand, the AE can not be stabilized between the double helix when the probe does not hybridize to the target. Under this condition, the ester linkage of AE undergoes hydrolysis. As a result, no chemiluminescence is generated and the target can not be detected: FIG. 2 (2).
The probe used in the detection may include, for example, probe 1 (SEQ ID NO: 3), probe 2 (SEQ ID NO: 4), probe 3 (SEQ ID NO: 5), probe 4 (SEQ ID NO: 6), probe 5 (SEQ ID NO: 7), probe 6 (SEQ ID NO: 8), probe 7 (SEQ ID NO: 9), and probe 8 (SEQ ID NO: 10). These probes 1 and 2, probes 3 and 4, probes 5 and 6, and probes 7 and 8 have the same sequence, respectively, but different binding sites of AE. The binding sites of AE were between 14th and 15th bases for probe 1, between 15th and 16th bases for probe 2, between 9th and 10th bases for probe 3, between 10th and 11th bases for probe 4, between 13th and 14th bases for probe 5, between 14th and 15th bases for probe 6, between 15th and 16th bases for probe 7, and between 16th and 17th bases for probe 8, respectively, in the respective base sequences.
The probe labelled with AE is added to the DNA or RNA amplified in the above manner (1) and incubated at 60xc2x0 C. for 5 to 30 minutes. To avoid chemiluminescence from unreacted probes, a hydrolysing reagent is added and incubated at 60xc2x0 C. for 5 to 10 minutes. After incubation, the chemiluminescence of AE is detected using a photomultiplier such as Reader I.
HPA using AE may also be carried out by using a kit from Gen-Probe Inc. according to the specification.
ii) Detection Using an Acridine Derivative or Other Non-radioactive Labelling Substance
To a solution containing the DNA or RNA, an appropriate amount of the probe labelled with the acridine derivative is added to react. After the reaction and subsequent hydrolysis, the chemiluminescence is detected as in the case of AE.
For luminol, isoluminol, pyrogallol, protohaemin, aminobutylethyl-n-isoluminol, and aminohexylethyl-n-ethyl-isoluminol, the above procedures or other methods may also be applied to detect chemiluminescence.
In detecting the telomerase activity according to the present invention, after an oligonucleotide sequence extended through the DNA extension reaction by a telomerase is amplified, the resulting amplified product can also be detected without using any radioactive labelling substance. For instance, a fluorescent substance such as FITC (Fluorescein Isothiocyanate), Rhodamine or Coumarin may be used to provide a fluorescent label at the 5xe2x80x2 end of the primer 1 (SEQ ID NO: 1). After electrophoresis, the amplified product can be detected by a given detector.
According to the method of the present invention, detection results can be obtained with a higher sensitivity as compared with conventional detection methods.
Further, since no radioactive substance is used, no special disposal equipment is required. Separation of the reaction products from non-incorporated radioactive substances as in the prior arts is not necessary. Consequently, the detection can be carried out rapidly. Thus, results can be obtained within one hour from the start of HPA operation; therefore, all detection processes may be completed within one day.
According to the present invention, telomerase activity can be easily detected with a good reproducibility and a large amount of sample may be dealt with.
The detection of telomerase activity in clinically obtained tissues or the like using the method of the present invention is usuful in the detection of cancer cells and diagnosis of cancer and is very useful in monitoring the progress of cancer or prognosis of treatment.
The cancer cells to be detected herein may include, for example, those contained in specimens obtained invasively or non-invasively.
The term xe2x80x9cinvasivelyxe2x80x9d used herein means that the collection of specimens is associated with bleeding when the specimens are taken by physically or chemically damaging or wounding human tissues or organs. For instance, examples of invasive methods include, for example, operation, excision of tissues by an endoscope, biopsy by a needle, and injection for exsanguinating. Specimens obtained invasively may include urinary bladder tissues, prostate gland tissues, uterus tissues, uterocervix tissues, udder tissues, pancreas tissues, liver tissues, large intestine tissues, stomach tissues, lung tissues, peripheral blood cells, kidney tissues, skin tissues, esophagus tissues, brain tissues and oral cavity tissues.
On the other hand, according to the present invention, specimens can be taken without physically or chemically damaging or wounding human tissues or organs; then, no bleeding is referred to as xe2x80x9cnon-invasivexe2x80x9d. For example, non-invasive methods may be exemplified by collection of excretions from bodies, and washing of organs. Specimens obtained non-invasively may include, for example, urine, prostate gland juice, urinary bladder wash, uterus smear, pancreatic juice, duodenum juice, feces, oral cavity wash, enteron wash, saliva and sputum.
(3) Kit for the Detection of Cancer Cells and/or the Diagnosis of Cancer
The kit of the present invention comprises a reagent used in the method for the detection of telomerase according to the present invention, that is, an oligonucleotide primer comprising at least a base sequence represented by xe2x80x9cAGNGTTxe2x80x9d wherein N is A, T, G or C at the 3xe2x80x2 end, an oligonucleotide primer comprising the base sequence represented by SEQ ID NO: 21 or an oligonucleotide primer comprising a base sequence having at least one nucleotide deleted, substituted or added in the base sequence of the primer, and a probe labelled with a non-radioactive labelling substance.
The kit of the present invention further comprises a primer having a promoter added to the 5xe2x80x2 end of said oligonucleotide primer.
These kits are used to detect the telomerase activity, to detect cancer cells, or to diagnose cancer.